Arsenic contamination of drinking water is a global health concern since exposure to arsenic has been linked with increased rates of cancer of skin, bladder, lung, liver and kidney. Arsenic is a known carcinogen; however the molecular mechanisms by which arsenic induces carcinogenesis remain undefined. Recent studies have shown that arsenite, an inorganic trivalent species of arsenic, induces oxidative stress when administered at clinically relevant concentrations in cell culture models. Selenium, a required micronutrient for mammalian cell growth and survival, is a component of several critical enzymes (thioredoxin reductase and glutathione peroxidases) with a role in defense against reactive oxygen species (ROS). Our laboratory has recently discovered that arsenite is a competitive inhibitor of selenophosphate synthetase, the first committed step in the pathway for insertion of selenium into selenoproteins. We have also determined that clinically relevant concentrations of arsenite inhibited the incorporation of selenium into selenoproteins in both keratinocytes (HaCat) and HeLa cells. Based on these observations, our hypothesis is that the increase in ROS observed in cells treated with arsenicals is due to inhibition of SPS and subsequent decreased levels of thioredoxin reductase (TrxR) and glutathione peroxidases (Gpx). We will test this hypothesis by: 1) Determining the effect of trivalent and pentavalent arsenicals on the incorporation of selenium into selenoproteins, 2.) Determining the level of thioredoxin reductase and glutathione peroxidase activity in HaCat cells treated with trivalent or pentavalent arsenicals while altering the nutritional source of selenium, and 3) Determining whether trivalent or pentavalent arsenicals inhibit either of the two human selenophosphate synthetase enzymes (SPS1 and SPS2) in vitro. The proposed experiments are designed to identify the arsenical(s) which affect selenoprotein synthesis and the molecular mechanism(s) involved. Relevance to Public Health: Arsenic is a known carcinogen, and selenium is an important micronutrient. We have evidence that certain arsenic compounds can block the synthesis of selenoproteins, which are important in the defense against oxidative damage that can lead to cancer. We believe arsenic acts to promote cancer by blocking the cell's ability to make selenoproteins. We will test this hypothesis and in doing so perhaps find selenium compounds that can counteract the negative effects of arsenic exposure. [unreadable] [unreadable] [unreadable]